Project 4 - SUMMARY Background: Secondary bacterial pneumonia caused by community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is the leading cause of death in patients following influenza A virus (IAV) infection. How IAV predisposes to severe CA-MRSA pneumonia is not fully understood. Hypothesis: We hypothesize that that a lethal synergism exists between influenza A neuraminidase (iNA) and S. aureus alpha- hemolysin (AH) that contributes to widespread lung destruction characteristic of this infection and that targeting the mechanism responsible for this synergy could lessen severity and improve outcomes in patients with post- influenza hemorrhagic necrotizing CA-MRSA pneumonia. Specific Aim 1: To determine the molecular mechanism whereby iNA augments AH-induced alveolo-capillary barrier destruction. For this, AH-induced ADAM10 protease activity in cultured pulmonary epithelial cells will be correlated with epithelial cell barrier structure and function in the presence and absence of iNA treatment. Specific Aim 2: To elucidate the role of iNA in priming the pulmonary microvascular endothelium for increased AH-induced platelet-neutrophil aggregate (PNA) deposition. PNAs contribute to tissue destruction in many soft tissue infections. We have shown that AH is the sole S. aureus exotoxin that mediates formation of PNA in human whole blood. To determine whether iNA augments this process, we will measure AH-induced PNA formation in whole blood and in the presence of pulmonary microvascular endothelial cells under static vs flow conditions with and without iNA treatment. Specific Aim 3. To assess the in vivo efficacy of an iNA inhibitor in decreasing the mortality associated with experimental post-influenza CA-MRSA pneumonia. Contrary to the current guidelines, we have shown the value of early anti-viral therapy, given alone, in improving outcome of experimental post- influenza MRSA pneumonia, whereas typical anti-MRSA antibiotics given alone were not efficacious. Using this model, we will investigate the mechanisms that underlay the efficacy of early vs late oseltamivir treatment (an iNA inhibitor), alone and together with antibiotics, in reducing lung destruction and improving outcome in post-influenza MRSA pneumonia. Impact on Human Health: Together, results from these studies may signal a paradigm shift in the current approach to treatment of post-influenza MRSA pneumonia. Contribution to Multi-disciplinary Infectious Diseases Research Program: Dr. Parimon blends clinical expertise in pulmonary medicine with a basic science understanding of the mechanisms of pathogenesis of severe bacterial pneumonia. Studies described in this proposal will increase the core's expertise pathogenic virology especially as it relates to post-influenza MRSA pneumonia. Because other bacterial soft tissue infections are purportedly made worse by antecedent viral infection, results from this study could be broadly applicable to several life-threatening diseases.